1. Field of the Invention
This invention relates generally to modified glycoprotein hormones. Specifically, this invention relates to modifications to a human glycoprotein which create superagonist activity.
2. Background Art
Thyrotropin (thyroid-stimulating hormone, TSH) and the gonadotropins chorionic gonadotropin, (CG), lutropin (luteinizing hormone, LH), and follitropin (follicle-stimulating hormone, FSH) comprise the family of glycoprotein hormones. Each hormone is a heterodimer of two non-covalently linked subunits: xcex1 and xcex2. Within the same species, the amino acid sequence of the xcex1-subunit is identical in all the hormones, whereas the sequence of the xcex2-subunit is hormone specific. (Pierce, J. G. and Parsons, T. F. xe2x80x9cGlycoprotein hormones: structure and function.xe2x80x9d Ann. Rev. Biochem. 50:465-495 (1981)). The fact that the sequences of the subunits are highly conserved from fish to mammals implies that these hormones have evolved from a common ancestral protein (Fontaine Y-A. and Burzawa-Gerard, E. xe2x80x9cEsquisse de I""evolution des hormones gonadotopes et thyreotropes des vertebres.xe2x80x9d Gen. Comp. Endocrinol. 32:341-347 (1977)). Evolutionary changes of these hormones resulted in certain cases in modification of biological activity (Licht, P. et al. xe2x80x9cEvolution of gonadotropin structure and function.xe2x80x9d Rec. Progr. Horm. Res., 33:169-248 (1977) and Combarnous, Y. xe2x80x9cMolecular basis of the specificity of binding of glycoprotein hormones to their receptors.xe2x80x9d Endocrine Rev. 13:670-691 (1992)), although, specific structural determinants modulating biopotency have not been elucidated. For example, human thyroid stimulating hormone (hTSH) and bovine thyroid stimulating hormone (bTSH) share high homology in the xcex1 (70%) and xcex2 (89%) subunit sequence, but bTSH is 6-10 fold more potent than hTSH (Yamazaki, K. et al. xe2x80x9cPotent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized thyroid hormone release in human thyroid follicles.xe2x80x9d J. Clin. Endocrinol. Metab. 80:473-479 (1995)).
Glycoprotein hormones are crucial in certain therapies, such as in the treatment of patients with thyroid carcinoma. (See, for example, Meier, C. A., et al., xe2x80x9cDiagnostic use of Recombinant Human Thyrotropin in Patients with Thyroid Carcinoma (Phase I/II Study).xe2x80x9d J. Clin. Endocrinol. Metabol. 78:22 (1994)). The potential use of human thyroid stimulating hormone (TSH) in the treatment of this disease has been abandoned due to the potential transmission of Creutzfeldt-Jakob disease. An alternative to the use of human TSH is the use of bovine TSH, but this approach is very limited since this hormone causes side-effects such as nausea, vomiting, local induration, urticaria, and a relatively high possibility of anaphylactic shock (Meier, C. A., et al.). The lack of bioconsistency of urinary gonadotropins and the limited efficacy of recombinant glycoprotein hormones justify their further replacement with more effective recombinant analogs. Therefore, there is a need for human-derived glycoprotein hormones as well as agonists of these hormones.
For example the administration of an agonist of the thyroid stimulating hormone in a particular clinical situation such as thyroid carcinoma, will enhance the uptake of radioiodine into the carcinoma to treat the disease. Agonists of the thyroid stimulating hormone will cause a greater amount of the radioiodine to be targeted to the carcinoma, thereby resulting in a more effective treatment. Alternatively, glycoprotein hormones used to induce ovulation can be replaced with superagonists. This will lower the required dose of the hormone which currently is a major medical problem in fertility treatment. (Ben-Rafael, Z., et al. xe2x80x9cPharmacokinetics of follicle-stimulating hormone: clinical significance.xe2x80x9d Fertility and Sterility 63:689 (1995)). Where the use of wild-type follicle stimulating hormone has led to hyperstimulation and higher rates of multiple pregnancies and abortions, apparently by a high number of hormone molecules stimulating many follicles, a superagonist of follicle-stimulating hormone can be administered to treat the infertility. The use of an agonist of this modified hormone can result in a lower frequency of stimulation of multiple follicles since a lower number of hormone molecules can be administered to achieve the desired result.
The present invention provides, for the first time, specific amino acid substitutions in human glycoprotein hormones which results in human glycoprotein hormone analogs that show a major increase in both in vitro and in vivo bioactivity.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, provides a human glycoprotein hormone comprising at least three basic amino acids in the xcex1-subunit at positions selected from the group consisting of positions 11, 13, 14, 16, 17 and 20.
The invention further provides a human glycoprotein hormone comprising at least one basic amino acid in the xcex1-subunit at positions selected from the group consisting of positions 11, 13, 14, 16, 17 and 20.
In another aspect, the invention provides a modified human glycoprotein hormone having increased activity over a wild-type human glycoprotein, wherein the modified human hormone comprises a basic amino acid substituted at a position corresponding to the same amino acid position in a non-human glycoprotein hormone having an increased activity over the wild-type human glycoprotein.
In another aspect, the invention provides a method of treating a condition associated with a glycoprotein hormone activity in a subject comprising administering a therapeutic amount of the glycoprotein hormone of the present invention to the patient.
In another aspect, the invention provides a method of constructing superactive nonchimeric analogs of human hormones comprising comparing the amino acid sequence of a more active homolog from another species to the human hormone, substituting amino acids in the human hormone with the corresponding amino acids from the other species, determining the activity of the substituted human hormone, and selecting superactive analogs from the substituted human hormones.
In yet another aspect, the present invention provides nucleic acids which encode the modified glycoprotein hormones.